1. Field of the Invention
The invention relates to novel nucleic acid assay reagents, a method for their preparation and their use for detecting and assaying nucleic acid sequences by hybridization techniques.
2. Brief Description of the Prior Art
The genetic information or code of all organisms, both eukaryotic and prokaryotic, consists of polymers of ribonucleotides (RNA) or deoxyribonucleotides (DNA). It is well known that complementary nucleotide molecules can interact by hydrogen bonding to form stable base-pairs. In this context, adenine recognizes and pairs with thymidine and guanine recognizes and pairs with cytosine. When two single-stranded, complementary nucleic acids are present in solution under the proper salt and temperature conditions, the complementary nucleotides can pair with one another and form a stable duplex or double-stranded structure. This phenomenon is termed "molecular hybridization" or "re-annealing" and underlies current methods for the detection of specific nucleotide sequences. In order to detect a specific nucleic acid sequence, a highly specific probe DNA or RNA sequence (which is complementary to all or part of the sequence to be determined) is isolated, amplified by cloning, purified to homogeneity and labeled with a suitable marker. For example, the purified probe DNA can be labeled with various marker chemicals and/or radioactive isotopes to facilitate the detection of duplex formation; see for example U.S. Pat. No. 4,599,303. The purified, labeled DNA is added to a hybridization solution containing denatured nucleic acids (RNA or DNA) from a sample to be tested. The aqueous conditions of the hybridization solution are adjusted to allow nucleic acid hybridization or reannealing, thereby allowing the labeled molecules to hybridize with unlabeled, complementary sequence counterparts. Duplex formation can be monitored by digestion with single strand-specific nucleases (such as S1 nuclease). Recovery and quantitation of resistant, i.e.; double-stranded, reannealed material provides a measure of the nucleic acid sequence tested for. The amount of hybridization is a function of the initial concentration of DNA and the time allowed for reannealing. Therefore, increased initial DNA concentrations can lead to substantially reduced hybridization times.
An example of the use of specific hybridization to detect sequences in nucleic acids is that described by Southern, E., J. Mol. Biol. 98:503, 1975. In this assay, a sample containing the DNA sequence to be detected is purified, digested with appropriate restriction endonucleases, and the fragments separated by gel electrophoresis. The fragments are then bound to a suitable solid support, such as nitrocellulose paper. This binding takes a minimum of 12-16 hours in the presence of a solution containing a relatively high concentration of sodium chloride. A labeled probe, complementary to sequences to be determined, is then added to the nitrocellulose paper and allowed to hybridize for a period of from 12 up to 48 hours. After this period of time, the paper must be washed under appropriate salt and temperature conditions since otherwise the labeled probe will bind non-specifically to both the paper and to other non-homologous DNA sequences, leading to background "noise" or "false positives". Indeed, one of the drawbacks of this assay is the difficulty in choosing the conditions for detecting the specific signal over the high background "noise". Another drawback of this assay is the time and labor involved in preparing the sample and performing the assay as well as in the hybridization and washing steps.
In a simplified version of the above-described Southern hybridization assay, nucleic acid samples to be analyzed are "dotted" onto a solid support (e.g. nitrocellulose or nylon filters) in an unfractionated state. The filters are then probed as in the Southern hybridization technique, washed, and the amount of bound probe is determined. This simplified format, called "dot hybridization," is less labor intensive than the Southern blot assay which requires that DNA samples be first restriction endonuclease digested, electrophoresed into gels, and transferred from the gels to the solid support. Even this simplified technique, however, suffers from the same problems in the choosing of the hybridization conditions, the time consumed, and the high background or high noise-to-signal ratio.
U.S. Pat. No. 4,563,419 to Ranki discloses a method for detecting nucleic acids using a one-step sandwich hybridization assay. The technique utilizes two nucleic probes each specific for non-overlapping sequences of DNA to be detected. Although this assay is said to be rapid and sensitive, the time required for hybridization is typically 16-20 hours. In addition, the presence of background (noise) due to the non-specific binding of DNA to the nitrocellulose filter is acknowledged.
U.S. Pat. No. 4,358,535 to Falkow et al. discloses a method for the detection of microbial pathogens based upon the use of DNA probes. The advantages of this assay include the ability to use clinical isolates without prior purification by classical bacteriological techniques. However, the assay has the same drawbacks as the above-mentioned prior art techniques for hybridization of complementary nucleic acids including background noise and time-labor requirements, due to the low concentrations of nucleic acids employed.
Obviously, what is needed is an assay for the detection of nucleic acid sequences that lends itself to standardization and overcomes or minimizes the above-mentioned drawbacks of currently used methods. The present invention meets this need and provides an assay which is versatile, rapid and has virtually none of the false positives or "noise" problems experienced in the assays of the prior art.
The assay of the present invention is flexible in that it may be modified to include many different assay samples in the same assay, has a low requirement for the quality of the DNA or RNA in the sample to be analyzed, exhibits sensitivity levels not available by other means and uses standardized reagents (except for the specific sequences of nucleic acid to be probed). In addition, the assay of the invention is suitable for use in conventional laboratory settings.